The globins and peroxidases, while performing completely different chemistry, share features of the iron heme active site: a protoporphyrin IX prosthetic group is linked to the protein by the proximal histidine residue. X-Ray absorption spectroscopy (XAS) provides a method to determine the local structure of iron heme active sites in proteins. Our previous studies using X-Ray absorption spectroscopy have revealed a significant difference in the Fe-N (e) bond length between the peroxidases and the globins (for a review, see Powers, L. in Molecular Electronics and Molecular Electronic Devices 3, 211: 1994, CRC Press Inc., Boca Raton, Florida). Globins typically have a Fe-N (e) distance close to 2.1 while the Fe-N (e) distance in the peroxidases is closer to 1.9. We have proposed (Sinclair, R., Powers, L., Bumpus, J., Albo, A., Brock, B. Biochem. 31: 4892, 1994 that strong hydrogen bonding to the proximal histidine is responsible for the shorter bond length in the peroxidases. Here we use site-specific mutagenesis to eliminate the strong proximal hydrogen bonding in cytochrome c peroxidase and to introduce strong proximal hydrogen bonding in myoglobin. Consistent with our hypothesis, elimination of the Asp-235-His hydrogen bond in CcP results in elongation of Fe-N (e) from ~1.9 to ~ 2.1. Conversely, introduction of a strong proximal hydrogen bond in myoglobin shortens Fe-N (e) from ~2.1 to ~1.9. These results correlate well with other biochemical data.